Biomimetic Design of Affinity Peptide Ligand for Capsomere of Virus

Jun 29, 2014 - rus (MPV) is a T = 7d icosahedral capsid that self-assembles from 72 ... It consists of 360 copies of major coat protein, VP1 (42 kDa),...
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Biomimetic Design of Affinity Peptide Ligand for Capsomere of Virus-Like Particle Yanying Li,† Xiaodan Liu,† Xiaoyan Dong,†,‡ Lin Zhang,*,†,‡ and Yan Sun*,†,‡ †

Department of Biochemical Engineering and Key Laboratory of Systems Bioengineering of the Ministry of Education, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, People’s Republic of China ‡ Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin 300072, People’s Republic of China S Supporting Information *

ABSTRACT: Virus-like particle (VLP) of murine polyomavirus (MPV) is a T = 7d icosahedral capsid that self-assembles from 72 capsomeres (Caps), each of which is a pentamer of major coat protein VP1. VLP has great potential in vaccinology, gene therapy, drug delivery, and materials science. However, its application is hindered by high cost downstream processes, leading to an urgent demand of a highly efficient affinity ligand for the separation and purification of Cap by affinity chromatography. Herein a biomimetic design strategy of an affinity peptide ligand of Cap has been developed on the basis of the binding structure of the C-terminus of minor coat protein (VP2-C) on the inner surface of Cap. The molecular interactions between VP2-C and Cap were first examined using all-atom molecular dynamics (MD) simulations coupled with the molecular mechanics/Poisson−Boltzmann surface area (MM/PBSA) method, where V283, P285, D286, W287, L289, and Y296 of VP2-C were identified as the hot spots. An affinity peptide library (DWXLXLXY, X denotes arbitrary amino acids except cysteine) was then constructed for virtual screening sequently by docking with AUTODOCK VINA, binding structure comparison, and final docking with ROSETTA FlexPepDock. Ten peptide candidates were selected and further confirmed by MD simulations and MM/PBSA, where DWDLRLLY was found to have the highest affinity to Cap. In DWDLRLLY, six residues are favorable for the binding, including W2, L4, L6 and Y8 inheriting from VP2-C, and R5 and L7 selected in the virtual screening. This confirms the high efficiency and accuracy of the biomimetic design strategy. DWDLRLLY was then experimentally validated by a one-step purification of Cap from crude cell lysate using affinity chromatography with the octapeptide immobilized on Sepharose gel. The purified Caps were observed to self-assemble into VLP with consistent structure of authentic MPV. expression level up to 4.38 g L−1 of VP1 with an N-terminal glutatione S-transferase (GST) tag has been achieved from the pH-stat fed-batch high-cell-density process in E. coli.9 While the fusion tag GST could enhance the soluble expression yield and improve the ease of tagged precursor purification, the subsequent enzymatic removal of GST with thrombin followed by isolation of tags and enzyme leads to high cost and obstacles, which present a daunting challenge in the production. Affinity chromatography is a highly selective separation technique that can be used to selectively purify a target molecule from a complex mixture by adsorption to specific ligands covalently attached to a resin matrix.10,11 The availability of ligand is crucial for affinity chromatography. Small peptide ligands have been in recent years a research focus for their specificity, stability and low lost.10,12,13 With the rapid development of molecular simulations in recent years, rational design has been extensively used in identifying highly selective peptide ligand against target protein, which is usually on the basis of the

1. INTRODUCTION Virus-like particles (VLPs) are highly organized nanoparticles that mimic the organization and conformation of authentic virus but are noninfectious due to the lack of the viral genetic material. Thus, VLPs have great potential in vaccinology, gene therapy, drug delivery, and materials science.1−4 Murine polyomavirus (MPV) VLP represents a striking model for a viral protein shell. It consists of 360 copies of major coat protein, VP1 (42 kDa), which self-assemble into capsomeres (Caps) positioned at a T = 7d lattice of approximate diameter of 40−50 nm.5 All Caps are pentamers of VP1, which are capable of occupying both pentavalent and hexavalent positions on the icosahedron due to the inter-Cap contacts formed by the flexible C-terminal invading arms that extend from each Cap into its neighbors.6 The VP1−VP1 interface interaction is a crucial factor that regulates the self-assembly process and determines whether the selfassembly biomolecules will be stable as Caps or associate to form either aggregates or capsids.4 VP1, purified after expression in Escherichia coli, can be obtained as Cap and self-assembles into VLP in a cell-free reactor.7,8 The in vitro assembly is an attractive strategy for the production of VLPs from a processing perspective. To date, © 2014 American Chemical Society

Received: May 6, 2014 Revised: June 21, 2014 Published: June 29, 2014 8500

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Scheme 1. Biomimetic Design and Screening of Affinity Peptide Ligands of Capa

a

(1) Analysis of the molecular interactions between the VP2-C and Cap. (2) Construction of the peptide library on the basis of the hot spots of VP2C. (3) Virtual screening by AUTODOCK VINA. (4) Comparing the distances between the atoms of the hot spots of VP2-C and those corresponding in the peptides by RMSD. (5) Selecting the peptides that have correct C-terminus orientation. (6) Final screening by ROSETTA FlexPepDock. (7) Validation of the affinity of candidate peptides using MD coupled with MM/PBSA method. (8) Experimental validation.

Figure 1. Initial conformation of the VP2-C/Cap complex for MD simulations: (a) bottom view, (b) side view of the interior surface. The VP2-C is shown in pink, and the three VP1 monomers that contact with VP2-C are in dark gray (chain A), cyan (chain B), and orange (chain E). The other two monomers are shown in iceblue (chain C) and lime green (chain D).

protein structures and the ligand-protein interactions that have already existed in natural world.14,15 Taking into account these affinity mechanisms in the design of peptide library could be more efficient and accurate, as proven in the literature.16−19 MPV internal protein VP2 (35 kDa) bound on the inner surface of Cap is an existing receptor-ligand complex. In vitro binding studies have confirmed that a 42-amino-acid fragment near the C-terminus of VP2 (thereafter termed VP2-C) is necessary and sufficient for the binding, with an estimated dissociation constant of 5 × 10−6 M.20,21 Crystal structure of VP2-Cap complex suggests that VP2-C inserts in an unusual, hairpin-like manner into the inner surface of Cap and binds strongly and specifically through hydrophobic interactions.22

In the present study, a biomimetic design strategy was proposed toward identifying high selective affinity ligand of Cap on the basis of a VP2−Cap complex as shown in Scheme 1. Molecular dynamics (MD) simulations coupled with the molecular mechanics/Poisson−Boltzmann surface area (MM/ PBSA) method4,18,19,23,24 were performed to explore the molecular interactions between VP2-C and Cap. On the basis of the affinity mechanism and the identified hot spots of VP2-C, a peptide library was built subject to virtual screening using AUTODOCK VINA,25 root-mean-square deviation (RMSD) comparison, orientation of C-terminus, and ROSETTA FlexPepDock.26 The affinities between the top 10 candidates and the target protein were then validated using MD and MM/ PBSA. Thereafter, the top one candidate was evaluated for its 8501

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solvation free energy (ΔGsol) was estimated as the sum of electrostatic solvation energy, calculated by the finite-difference solution of the Poisson-Boltzmann equation (ΔGPB) using the PBEQ module of the CHARMM program and nonpolar solvation free energy (ΔGnp), calculated from the solvent-accessible surface area (SASA). The solute and solvent dielectric constants were set to 1 and 80, respectively, in the calculation of ΔGPB. ΔGnp, which could be considered as the sum of a solvent−solvent cavity term and a solute-solvent vdW term, was calculated according to eq 4, where the constants γ and b were set to 0.00542 kcal/(mol.Å2) and 0.92 kcal/mol, respectively.40 The SASA was calculated using a water probe radius of 1.4 Å. In free energy decomposition, the free energy contribution of each residue can be divided into polar (ΔGpolar) and nonpolar parts (ΔGnonpolar) according to eq 5, and each part is the sum of two energy terms, as given in eqs 6 and 7. In the following analysis, ΔGpolar is considered as the contribution by electrostatic interaction and ΔGnonpolar as the contribution by hydrophobic interaction.4

potential to selectively recognize Cap and serve as ligand for affinity chromatography of Cap.

2. MODELS AND METHODS 2.1. Model Construction. Coordinate of VP2-C/Cap complex was taken from the crystal structure from the Protein Data Bank (PDB ID: 1CN3; http://www.rcsb.org/pdb/)22 as shown in Figure 1. The VP2-C (residues from 279 to 297) is located along the inner surface of Cap and contacts with three VP1 monomers. These residues form an ordered stretch followed by a sharp 90° blend at P285, leading to a two-turn αhelix.22 For Cap, residues from 34 to 316 were used. Lysine and arginine were positively charged with a charge number of +1 each, while glutamic acid and aspartic acid were negatively charged with a charge number of −1 each, leading to a total charge number of −35 in the complex (pH 7.0). The complex was then solvated in Stabilization Buffer (200 mM NaCl),4 where the water molecule was treated using the TIP3P model, and Na+ and Cl− were considered as charged beads. To prepare the simulation box, the VP2-C/Cap complex was first placed in the center of a cubic box (12.16 × 12.16 × 12.16 nm3) filled by water molecules. Then 225 Na+ and 190 Cl− were added by replacing the corresponding number of water molecules according to the NaCl concentration and the neutralization of the simulation system. 2.2. Molecular Dynamics Simulations. MD has been widely used to examine protein behaviors at interfaces, especially the molecular interactions and orientation revealed in binding processes.27−32 In this work, MD simulations were performed using the GROMACS 4.5.3 package (http://www.gromacs.org/)33 with an all-atom CHARMM27 force field. Prior to simulation, each system was subject to 10 000 steps of steepest descent energy minimization. Then the system was equilibrated for 200 ps under NVT ensemble and another 200 ps under NPT ensemble. Thereafter, MD simulations were carried out under NPT ensemble for 20 ns. Temperature was controlled at 298.15 K4 by the velocity-rescale (v-rescale) method34 with a time constant of 0.1 ps. The pressure was controlled at 1 atm by Parrinello−Rahman with a coupling constant of 2 ps. Newton’s equations of motion were integrated using the leapfrog algorithm with a 2 fs time step. The shortrange van der Waals interactions were cut off at 1.2 nm. The long-range electrostatic interactions were treated with the particle mesh Ewald (PME) method,35 with the real-space contribution to the Coulombic interactions truncated at 1.2 nm. All bonds were constrained with the LINCS algorithm36 with a relative geometric tolerance of 10−4. Initial velocities were assigned according to a Maxwell distribution. All simulations were performed on a 160 CPU Dawning TC2600 blade server (Dawning, Tianjin, China). 2.3. Binding Free Energy Analysis. The binding free energies (ΔGbind) were calculated using the MM/PBSA method4,18,19,23,37 and CHARMM38 program (http://www.charmm-gui.org/), following the procedure reported previously4,18,19,37 with minor modification. 76 snapshots were extracted from the last 3 ns trajectory of simulation performed by GROMACS at an interval of 40 ps. The binding free energy (ΔGbind) is calculated according to the following equations:

ΔG bind = ΔGgas + ΔGsol − T ΔS

(1)

ΔGgas = ΔGelec + ΔGvdW

(2)

ΔGsol = ΔG PB + ΔGnp

(3)

Gnp = γ × SASA + b

(4)

ΔG bind = ΔGpolar + ΔGnonpolar

(5)

ΔGpolar = ΔGelec + ΔG PB

(6)

ΔGnonpolar = ΔGvdW + ΔGnp

(7)

2.4. Biomimetic Design of Affinity Ligand. On the basis of the affinity mechanism and the hot spots identified above, a peptide library of DWXLXLXY was designed and constructed using our in-house scripts with the program embedded in the CHARMM package38 (http://www.charmm-gui.org/), where X stands for 19 arbitrary amino acid residues except for cysteine (cysteine was not considered herein to avoid intramolecular cyclization or intermolecular disulfide formation). Then, AUTODOCK VINA 1.1.225 (http://vina.scripps.edu/) hereafter termed VINAwas used to dock all peptides against Cap. The crystal structure of Cap (PDB ID: 1CN3) was used as the receptor for docking while the candidates in the designed peptide library were used as the ligands. Prior to docking, a PBDQT file for the receptor and ligands were prepared using AUTODOCKTOOLS 1.5.4.25,41 Default parameters were used as described in the manual of VINA25,41 unless otherwise specified. The affinity energy value (kcal/mol) for each ligand was calculated and denoted by EVINA. For each ligand candidate, the best docked conformation was chosen for the following analysis. The auxiliary program g_rms provided by the GROMACS 4.5.3 package33 (http://www.gromacs.org/) was used to evaluate the structural deviation between the atoms of the hot spots of VP2-C and those corresponding in the peptides. The coordinates of the peptides were obtained from VINA docking while the coordinates of Cap was obtained from the crystal structure of the VP2-C/Cap complex. Each candidate DWXLXLXY contains five key residues. The conformation of these residues may be changed during the docking. Then RMSD of these residues was calculated with respect to the original structure in the VP2C to provide a quantitative evaluation of the change. Small RMSD indicates a similar binding structure of this candidate as that in the VP2C. Furthermore, the binding sites of each candidate on Cap were checked as compared with the binding sites of VP2-C. Any candidate without similar binding sites was not considered in the following screening. Thereafter, the ROSETTA FlexPepDock web server26,42 (http:// flexpepdock.furmanlab.cs.huji.ac.il/) was used to rescreen the candidate peptides selected from VINA docking and RMSD comparison. FlexPepDock is a high-resolution peptide docking (refinement) protocol, implemented within the ROSETTA framework. The initial conformation of the protein-peptide complex was derived from the results of VINA docking. The initial structure was refined in 200 independent FlexPepDock simulations, producing 100 low-resolution and 100 high-resolution structures. Then the interface energy score (I_sc)19 was calculated for evaluating the binding affinity of each candidate on the Cap. 2.5. Synthesis of Peptide Resin and DWDLRLLY-Cap Binding Assay. For synthesis of peptide resin, the candidate ligand DWDLRLLY was extended at the C-terminus to include a cysteine residue (DWDLRLLYC) for coupling by GL Biochem (Shanghai,

where ΔGgas is the gas-phase energy; ΔGsol, the salvation energy; −TS, the entropic energy. The brackets, ⟨···⟩, indicate an average of an energy term along the MD simulation trajectory. In this study, the entropy contribution was not estimated because the major objective was to identify residues that play a dominant role in stabilizing the complex rather than to obtain a quantitative account of the binding thermodynamics. Due to its high computational demand and negligible change as compared with ΔGbind, entropy contribution was excluded in the free energy calculation as that in recent literature39 and our previous work.4,18,19,37 ΔGgas is the sum of ΔGelec and ΔGvdW (eq 2). The 8502

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China) and conjugated to Thiopropyl Sepharose 6B (GE Healthcare, Uppsala, Sweden) according to the manufacturer instruction. Ligand density was determined by analyzing the reaction supernatant before and after coupling by analytical reversed phase chromatography using a C18 reversed-phase column (Milford, MA, USA) (150 mm × 4.6 mm I.D., 5 μm particle size) on Agilent 1100 series (Agilent Technologies, Santa Clara, California, USA). As a result, no peptide was detected in the supernatant after coupling as illustrated in Figure S1. To investigate the ability of the peptide resin to recognize Cap, 0.1 g pre-equilibrated adsorbent (50 mM PBS, pH 6.0, containing 0.2 M NaCl) was added to a 25 mL Erlenmeyer flasks containing 5 mL clarified cell lysate (please see the detailed protocols in the Supporting Information) and then incubated at 25 °C, 170 rpm for 24 h to achieve adsorption equilibrium (This time was confirmed to allow the adsorption to reach equilibrium by preliminary experiments). The mixture was centrifuged at 10 000 rpm for 1 min, and the supernatant was taken for SDS-PAGE analyses. For comparison, blank resin with no peptide attached was also used as the control. 2.6. Peptide Affinity Column Purification of Cap from Cell Lysate. Chromatographic experiment was performed on Ä KTA FPLC (GE Healthcare) with a HR5/5 column (GE Healthcare) packed with peptide resins. The absorbance was detected at 280 nm using an external UV detector (GE Healthcare). The clarified cell lysate was used as the feedstock for the affinity purification of Cap. Fifty milimolar PBS buffer, pH 7.0, containing 0.2 M NaCl was used for equilibration and washing the column after loading 200 μL of clarified cell lysate. The bound protein was eluted with 50 mM citrate buffer, pH 3.0, and the regeneration was carried out by 100 mM Gly-HCl, pH 2.4. Washing, elution, and regeneration were all performed at the flow rate of 0.5 mL/ min (153 cm/h) while a linear velocity of 60 cm/h was applied for injection. Protein samples in the feedstock, flow through fraction and the eluted fraction were collected, concentrated, and subject to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The electrophoresis image was analyzed by the software Gel-Pro Analyzer 3.1 (Media Cybernetics, MD) to determine the purity. 2.7. VLP Assembly. Assembly of purified Caps to VLP was induced by dialyzing Caps in Assembly Buffer 1 (0.5 M (NH4)2SO4, 20 mM Tris, 5% (v/v) glycerol, 1 mM CaCl2, pH 7.4) for 15 h, and then against Assembly Buffer 2 (200 mM NaCl, 20 mM Tris, 5% (v/v) glycerol, 1 mM CaCl2, pH 7.4) for another 24 h according to the literature.7 Thereafter, 10 μL aliquots of VLP preparations were placed to glowdischarged, 200-mesh carbon-coated copper grids (Xinxing Braim Technology Co., Ltd., Beijing, China) and allowed to adsorb for 2 min. Samples were then stained with 2% (w/w) phosphotungstic acid (pH 7.4) for 15 min and exampled with a JEM-2100F (Jeol Ltd., Tokyo, Japan) microscope at 200 kV.

Figure 2. RMSD of Cap and VP2-C (top) and the intermolecular potential energy between VP2-C and Cap (bottom) as a function of simulation time. RMSD of heavy atoms from the initial structure is computed.

Table 1. Binding Free Energies (kcal/mol) of VP2-C/Cap Complex energy term

energy (kcal/mol)

ΔGvdw ΔGelec ΔGPB ΔGnp ΔGnonpolara ΔGpolarb ΔGbindc

−69 ± 5 −171 ± 15 174 ± 17 −10 ± 0.3 −79 ± 5 3±8 −76 ± 8

ΔGnonpolar = ΔGvdW + ΔGnp, hydrophobic interaction energy. bΔGpolar = ΔGelec + ΔGPB, electrostatic interaction energy. cΔGbind = Gnonpolar + ΔGpolar. a

driving force for the binding of VP2-C on the inner surface of Cap is hydrophobic interaction rather than the electrostatic interaction, which is in line with experimental fact in terms of crystal structure.22 3.2. Identification of Hot Spots for VP2-C/Cap Complex and Construction of VP2-Mimic Peptide Library. The hot spots that make significant contributions for the VP2-C/Cap complex were identified as illustrated in Figure 3, using a criterion of ±2.5 kcal/mol.44 For Cap, six residues are identified as hot spots, including five residues (K126, N257, T258, T260, and R311) in chain A and one residue (R238) in chain B. The spatial distribution of these residues is illustrated in Figure S2. For VP2-C, six residues (V283, P285, D286, W287, L289, and Y296) located around the inner surface of Cap are found to have great contributions to the binding. Five residues are favorable for binding, while D286 is strongly unfavorable with a positive ΔGbind of 3.6 kcal/mol. Pair interaction analysis was conducted to detect the important interactions of VP2-C/Cap complex (Figure S3, Supporting Information). Obviously, all of the above-mentioned residues contributing large favorable binding free energies are involved in the formation of strong intermolecular interactions. According to the spatial distribution of hot spots of VP2-C (Figure 4a), a binding motif was constructed (Figure 4b), where the residues making dominant contributions to binding are represented by yellow balls, while the ones having minor contributions are represented by red balls. It should be noted that although D286 seems to make an unfavorable contribution for the binding (Figure 3), it can form a salt bridge/hydrogen bond

3. RESULTS AND DISCUSSION 3.1. Binding Free Energy Calculation Analysis. It has been proven by extensive studies4,18,19,23,37,43 that the relative binding free energy calculated using MD simulations coupled with the MM/PBSA method can provide successful evaluation of affinities between protein molecules. Therefore, MD runs of 20 ns were performed at first to generate a set of ensembles of conformations. Then RMSD and intermolecular potential energies were calculated. As shown in Figure 2, the intermolecular potential energies indicate that the complex has been well equilibrated. The RMSD profiles of both VP2-C and Cap reach equilibrium at a time scale of 5 ns. Therefore, the coordinates generated in the last 3 ns of the trajectories were collected for the calculation of relative binding free energy using MM/PBSA method. As shown in Table 1, the total binding free energy (ΔGbind) of VP2-C/Cap complex is -76.31 kcal/mol, which can be further decomposed into a nonpolar part (ΔGnonpolar) of −79.28 kcal/ mol and a polar part (ΔGpolar) of 2.98 kcal/mol. Larger absolute value of ΔGnonpolar than that of ΔGpolar indicates that the major 8503

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that L293 is aligned in a straight line with W287, L289 and Y296, which is favorable for the design of short linear peptides without consideration of the spatial structures of VP2. Moreover, it was reported that the mutation L181E on simian virus 40 (SV40) minor coat protein VP3 causes reduction of Cap binding to 33%,45 where L181 in SV40 VP3 is just the counterpart of L293 in MPV VP2 herein. Therefore, in consideration of the high conserved region in polyomaviruses,45 L293 is considered to be important in the binding of VP2-C/Cap complex and thus included in the binding motif. Therefore, the binding motif of VP2-C is composed of V283, P285, D286, W287, L289, L293 and Y296 (Figure 4b). All seven of the residues in the binding motif can be used for library design. However, including all seven residues in the library design causes a huge number of candidates up to millions, which is impossible for screening. Therefore, D286, W287, L289, L293, and Y296 located at the helix were selected for library design. The distance between these residues is labeled in Figure 4b. In order to reproduce the affinity between VP2 and Cap, the spatial arrangement of these residues should be guaranteed to be in accordance with that of VP2. It is known that the lengths of a peptide bond and an amino acid backbone are about 1.33 and 2.78 Å, respectively. According to the distances among these amino acid residues (W287−L289 = 5.42 Å, L289−L293 = 5.72 Å, L293−Y296 = 5.61 Å) marked in Figure 4b, one residue can be inserted between every two adjacent residues. Thus, an octapeptide library of DWXLXLXY containing 6859 peptides was rationally designed on the basis of the binding motif proposed above, where “X” represents 19 arbitrary amino acid residues (except cysteine, as indicated in Section 2.4). 3.3. Screening of Peptide Library Using Molecular Docking. In order to evaluate the affinity between peptides and Cap, 6859 peptides were first docked into the target pocket located on the inner surface of Cap using VINA and ranked according to the EVINA, as shown in Figure S5. The EVINA ranges from −3.9 to −7.7 kcal/mol, indicating widespread candidates in the library. Then, according to the distribution of EVINA, 1158 peptide candidates with EVINA ≤ −6.5 kcal/mol were selected subjected to following screening. For a good candidate, its docked conformation should mimic the binding structure in VP2-C/Cap complex. Therefore, the structure similarity has been evaluated by RMSD, where the original structure in the VP2-C was chosen as the reference structure. Then small RMSD value indicates high structure similarity of the docked conformation to VP2-C/Cap complex. The distribution of RMSD is shown in Figure S6, and 327 peptides with RMSD values of less than 0.4 nm were selected for the next screening. In the VP2-C/Cap complex, the VP2-C runs from the bottom to the top of the inner surface of Cap, and the C-terminus is located at the bottom of Cap. Then the peptides without similar orientation with VP2-C, e.g., with N-terminus toward the bottom of Cap, were not considered in the following screening. Then 227 peptides were selected for the next screening. The selected peptides were then rescreened by flexible molecular docking using ROSETTA FlexPepDock. The top 10 peptides with the highest absolute I_sc values are listed in Table S1. It can be seen that DWDLRLLY shows the highest absolute value of I_sc. The top 10 peptides with high score are considered as potential highly efficient affinity ligands. 3.4. Validation by MD Simulations and MM/PBSA. Ten potential high efficient affinity ligands were then validated using

Figure 3. Binding free energy contribution of each residue in VP2-C and Cap in the VP2-C/Cap complex. Residues with |ΔGbind| ≥ 0.5 kcal/mol are shown. The residue is colored by its contribution to binding free energy. The error bars indicate the standard deviations.

Figure 4. Construction of the affinity binding model of VP2-C. The hot spots of VP2-C are colored according to the free energy contribution of each residue (a). Binding free energy ranges from red (most negative) to blue (most positive). A simplified binding motif of VP2-C is given in (b), where the residues making dominant contributions to binding are represented by yellow balls while the ones having minor contributions are represented by red balls. The distances between residues (in angstroms) are marked in purple. The figures were prepared using the VMD software (http://www.ks.uiuc.edu/Research/vmd/).

network with K126 and T260 of chain A and with R238 of chain B.22 It is observed from Figure S3 that D286 of VP2-C interacts with R238-B, R313-A, and R311-A via favorable electrostatic interaction. However, D286 has unfavorable electrostatic repulsion with E266-B, E266-A, and E121-A, simultaneously, leading to the final unfavorable contribution with a positive ΔGbind of 3.6 kcal/mol. Then a mutant D286A of VP2-C was constructed subject to MD simulation and analysis of molecular interaction energies (Figure S4). Significant reduction of electrostatic interaction energy is observed, indicating that D286 does play an important role in the binding between Cap and VP2-C. Therefore, D286 was identified as hot spot and included in the binding motif. Meanwhile, it should be noted that the contribution of L293 to the binding free energy is not satisfied with the criterion of hot spots. However, it can be seen 8504

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Table 2. Binding Free Energies of the Top 10 Potential Ligand/Receptor Complexes

a

no.a

peptide

ΔGbind

ΔGnonpolar

ΔGpolar

1 2 3 4 5 6 7 8 9 10 Model

DWDLRLLY DWDLRLIY DWNLRLIY DWFLNLFY DWSLKLVY DWSLRLKY DWNLHLPY DWSLDLWY DWGLRLKY DWGLKLIY VP2-C

−61.01 ± 7.50 −54.94 ± 8.48 −48.07 ± 5.43 −37.39 ± 8.42 −37.03 ± 11.34 −23.83 ± 8.95 −20.86 ± 8.43 −19.25 ± 6.43 −14.30 ± 4.63 −6.40 ± 4.84 −76.31 ± 8.40

−55.44 ± 4.39 −54.63 ± 6.03 −53.13 ± 3.37 −75.68 ± 4.08 −49.25 ± 5.23 −50.00 ± 7.22 −39.19 ± 7.24 −38.99 ± 5.14 −22.28 ± 4.16 −15.21 ± 3.60 −79.28 ± 4.80

−5.58 ± 8.93 −0.31 ± 10.35 5.06 ± 4.95 38.31 ± 9.39 12.22 ± 14.10 26.18 ± 12.58 18.33 ± 7.26 19.75 ± 8.42 7.98 ± 2.33 8.82 ± 5.10 2.98 ± 8.30

The peptides are ranked by the ΔGbind value.

Figure 5. (a) Binding free energy contribution of each residue within DWDLRLLY in the DWDLRLLY/Cap complex as compared with the hot spots in VP2-C. (b) The comparison of the spatial position between residues on DWDLRLLY and their corresponding hot spots on the VP2-C.

position that is supposed to belong to D1, which has a role similar to that of D286 in VP2-C. The above results demonstrate the high efficiency and accuracy of the biomimetic design strategy. For further in-depth discussion, the binding free energy contribution of each residue within Cap in the DWDLRLLY/ Cap complex is compared with that in VP2-C/Cap complex (Figure 6). Eleven residues have been observed in DWDLRLLY/ Cap but not yet in VP2-C/Cap, and most of them are favorable for binding. The favorable binding free energy (ΔGbind,−) is −12 kcal/mol, which is close to that of only observed in VP2-C (-13 kcal/mol). Twelve residues are observed in both VP2-C/Cap and DWDLRLLY/Cap, five of which are hot spots in VP2-C/ Cap complex. Most of the hot spots also make significant contributions for the binding between DWDLRLLY and Cap, especially the K126-A and T260-A. It is worth to pointing out that hot spots of Cap are mainly located on one monomeric VP1 (chain A), indicating that the DWDLRLLY may also have affinity for monomer VP1 that exists in free form. Therefore, DWDLRLLY has been confirmed to capture the binding nature of VP2-C/Cap complex and thus an efficient affinity ligand for Cap. Further experimental validations are then performed on DWDLRLLY to confirm its affinity and specificity to Cap. 3.5. Experimental Validation. DWDLRLLY was coupled onto Thiopropyl Sepharose 6B at a ligand density of 2 μmol/(g drained wet resins) (thereafter termed Pep-6B) to investigate its ligand-binding nature. The blank resin was used as the control.

MD simulations coupled with MM/PBSA. It is shown that except DWGLKLIY, DWGLRLKY and DWSLDLWY, the other seven peptides kept a stable binding conformation with the inner surface of Cap during the MD simulation and thereby were predicated to have high affinity with Cap (Figure S7). The binding free energies of these peptides on the inner surface of Cap were calculated by MM/PBSA, and the results are listed in Table 2. For all peptides, the nonpolar contributions is dominate rather than the polar contributions, which is in good agreement with the VP2-C/Cap complex, demonstrating the accuracy of the rational design strategy. The lowest total binding free energy of these peptides is observed in DWDLRLLY-Cap (ΔGbind = −61 kcal/mol), close to ΔGbind (−76 kcal/mol) for the VP2-C/Cap complex. Thereby DWDLRLLY is considered as a good candidate ligand with high affinity to Cap. The free energy decomposition results of DWDLRLLY are presented in Figure 5a. According to the criterion of hot spot, seven residues (W2, D3, L4, R5, L6, L7, and Y8) of DWDLRLLY are identified as hot spots, among which L4, L6, and Y8 inherit from VP2-C and provide enhanced favorable contribution for the binding as indicated by more negative value of ΔGbind than their counterparts in VP2-C; R5 and L7 selected in virtual screening have negative values of ΔGbind, providing further enhancement of binding affinity to Cap. Moreover, in DWDLRLLY, spatial arrangement of the hot spots similar to that of their counterparts in VP2-C (Figure 5b) is observed. Although D1 is far from its corresponding D286 in VP2-C, another Asp (D3) replaces the 8505

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Pep-6B column successfully by decreasing ionic strength and pH. That is because decreasing the ionic strength would weaken the hydrophobic interaction and reduce the affinity, further confirming the favorable contribution of hydrophobic interaction for binding. Additionally, the theoretical pI of DWDLRLLY is 4.21, while that of Cap is 6.0, as calculated by the ExPASy ProtParam (http://web.expasy.org/protparam/). Then both the peptide and Cap are positively charged in elution buffer (50 mM citrate buffer, pH 3.0), leading to electrostatic repulsion, which could assist the elution of Cap from Pep-6B. The eluted peak (lane 3) contains mainly VP1 with minor impurities. Analysis of the SDS-PAGE image by Gel-Pro Analyzer reveals that Pep-6B dramatically improves the purity of VP1 from 15.6% (lane 1) to 70.1% (lane 3). However, it should be noted that some impurities are still retained in the elution fraction (lane 3), which can be attributed to nonspecific adsorption driven by hydrophobic interaction caused by nonpolar residues in DWDLRLLY. Since the separation procedure has not been optimized to any extent, it is anticipated that the optimization of operating parameters, such as the ligand density, modification of the washing and elution process would improve the separation performance of the peptide affinity chromatography. Anyway, the present result has further confirmed the high affinity and specificity of the Pep-6B for Cap. The self-assembly of purified Cap to VLP was then induced by dialysis of purified Cap against Assembly Buffer and analyzed by transmission electron micrograph (TEM). As shown in Figure 9, the VLP obtained is homogeneous in size with a diameter ranging from 40 to 50 nm, which is comparable to that of authentic MPV,46 indicating the basic functional characteristics of Cap. Therefore, the aforementioned results demonstrate the feasibility of biomimetic design to screen highly selective peptide ligand for Cap.

Figure 6. Binding free energy contribution of each residue within Cap in the DWDLRLLY/Cap complex as compared with that in VP2-C/Cap complex. The residues with |ΔGbind| ≥ 0.5 kcal/mol are shown. These residues are divided into three parts, i.e., residues only observed in DWDLRLLY/Cap, residues observed in both DWDLRLLY/Cap and VP2-C/Cap, and residues only observed in VP2-C/Cap. In each part, the residues are further divided into two clusters, including one favorable for binding and the other unfavorable for binding. The sum of the binding free energy in each cluster is calculated and provided in the figure.

As illustrated in Figure 7, almost all Caps in the clarified cell lysate bind to the Pep-6B since no monomeric VP1 is observed in

4. CONCLUSIONS Discovery of a highly efficient affinity ligand for the separation and purification of Cap is of great significance for low-cost downstream processes of VLP. In the present study, a biomimetic design strategy for affinity peptide ligands of Cap was developed on the basis of the binding structure of VP2-C on the inner surface of Cap. Molecular mechanism of the affinity between VP2-C and Cap was explored using MD simulations coupled with MM/PBSA analysis. Hydrophobic interaction is found as the major driving force, and residues V283, P285, D286, W287, L289, and Y296 of VP2-C are identified as the hot spots, leading to the design of novel VP2-mimic ligands of Cap through the construction of a peptide library of DWXLXLXY. DWDLRLLY selected by the virtual screening of peptide library was found to have the highest affinity for Cap, which are further confirmed by MD simulations and MM/PBSA analysis. Six residues in DWDLRLLY are confirmed to be favorable for the binding, including W2, L4, L6, and Y8 inheriting from VP2-C, R5 and L7 selected in virtual screening. This confirms that the biomimetic design strategy is efficient and accurate for the screening of affinity peptide ligands of Cap. Finally, the affinity and specificity of DWDLRLLY to Cap was experimentally validated by binding assay and one-step purification of Cap from cell lysate using affinity chromatography. Therefore, it is expected that the biomimetic design strategy would greatly facilitate the production and application of VLP. Further work can direct toward optimization of some key parameters, such as ligand density and washing and elution conditions, to improve the purification performance. Meanwhile, the other six

Figure 7. SDS-PAGE analysis of DWDLRLLY-Cap binding assay. Lane 1: clarified cell lysate; Lane 2: blank resin adsorption supernatant; Lane 3: peptide resin adsorption supernatant; M: molecular weight markers.

lane 3. In contrast, the Cap cannot be adsorbed to the blank resin (lane 2). At the same time, most contaminants are retained in the peptide resin adsorption supernatant (lane 3), demonstrating that Pep-6B can selectively recognize Cap from a heterogeneous mixture of proteins. Adsorption at relatively high ionic strength (Stabilization Buffer: 0.2 M NaCl) suggests the domination role of hydrophobic interaction between DWDLRLLY and Cap, which is in accordance with the simulation results and experimental fact reported earlier.22 To further examine the specificity and affinity of DWDLRLLY, affinity chromatography was used for the purification of Cap from clarified cell lysate. The typical chromatogram of the affinity chromatography is shown in Figure 8a while the feedstock, flowthrough fraction and eluted fraction together with the regeneration fraction were analyzed by SDS-PAGE as illustrated in Figure 8b. It is clear that almost all Caps bind to the Pep-6B column since no monomeric VP1 is observed in the flow-through fraction (lane 2). Moreover, most Caps can be eluted off from the 8506

dx.doi.org/10.1021/la5017438 | Langmuir 2014, 30, 8500−8508

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Figure 8. (a) Typical elution profile for Cap purification from clarified cell lysate. Detection at 280 nm. (b) SDS-PAGE analysis. Lane 1: feedstock; Lane 2: flow-through fraction; Lane 3: elution fraction; Lane 4: regeneration fraction; Lane 5: pure VP1; M: molecular weight markers.

Doctoral Program of Higher Education (SRFDP) (No. 20130032110028), and the Innovation Foundation of Tianjin University.

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Figure 9. TEM of VLP that self-assembles from purified Caps. Scale bar is 100 nm.

octapeptides identified in the virtual screening can be experimentally evaluated. Moreover, some residues with little favorable or even unfavorable contributions for the binding (e.g., D1) can be replaced by other amino acid residues to improve the affinity and discover more effective ligands for Cap.

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ASSOCIATED CONTENT

S Supporting Information *

Cloning, expression and preparation of Cap in E. coli, as well as additional figures and tables. This material is available free of charge via the Internet at http://pubs.acs.org.

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REFERENCES

(1) Salunke, D. M.; Caspar, D. L. D.; Garcea, R. L. Self-assembly of purified polyomavirus capsid protein VP1. Cell. 1986, 46 (6), 895−904. (2) Ghasparian, A.; Riedel, T.; Koomullil, J.; Moehle, K.; Gorba, C.; Svergun, D. I.; Perriman, A. W.; Mann, S.; Tamborrini, M.; Pluschke, G. Engineered synthetic virus-like particles and their use in vaccine delivery. ChemBioChem 2011, 12 (1), 100−109. (3) Uchida, M.; Morris, D. S.; Kang, S.; Jolley, C. C.; Lucon, J.; Liepold, L. O.; LaFrance, B.; Prevelige, P. E., Jr.; Douglas, T. Site-directed coordination chemistry with P22 virus-like particles. Langmuir 2011, 28 (4), 1998−2006. (4) Zhang, L.; Tang, R.; Bai, S.; Connors, N. K.; Lua, L. H.; Chuan, Y. P.; Middelberg, A. P.; Sun, Y. Molecular energetics in the capsomere of virus-like particle revealed by molecular dynamics simulations. J. Phys. Chem. B 2013, 117 (18), 5411−5421. (5) Stehle, T.; Yan, Y.; Benjamin, T. L.; Harrison, S. C. Structure of murine polyomavirus complexed with an oligosaccharide receptor fragment. Nature 1994, 369, 160−163. (6) Ding, Y.; Chuan, Y. P.; He, L.; Middelberg, A. P. J. Modeling the competition between aggregation and self-assembly during virus-like particle processing. Biotechnol. Bioeng. 2010, 107 (3), 550−560. (7) Chuan, Y. P.; Lua, L. H. L.; Middelberg, A. P. J. High-level expression of soluble viral structural protein in Escherichia coli. J. Biotechnol. 2008, 134 (1), 64−71. (8) Pattenden, L. K.; Middelberg, A. P. J.; Niebert, M.; Lipin, D. I. Towards the preparative and large-scale precision manufacture of viruslike particles. Trends Biotechnol. 2005, 23 (10), 523−529. (9) Liew, M. W. O.; Rajendran, A.; Middelberg, A. P. J. Microbial production of virus-like particle vaccine protein at gram-per-litre levels. J. Biotechnol. 2010, 150 (2), 224−231. (10) Kaufman, D. B.; Hentsch, M. E.; Baumbach, G. A.; Buettner, J. A.; Dadd, C. A.; Huang, P. Y.; Hammond, D. J.; Carbonell, R. G. Affinity purification of fibrinogen using a ligand from a peptide library. Biotechnol. Bioeng. 2002, 77 (3), 278−289. (11) Vutukuru, S.; Kane, R. S. Surface plasmon resonance spectroscopy-based high-throughput screening of ligands for use in affinity and displacement chromatography. Langmuir 2008, 24 (20), 11784−11789. (12) Makriyannis, T.; Clonis, Y. Design and study of peptide-ligand affinity chromatography adsorbents: Application to the case of trypsin purification from bovine pancreas. Biotechnol. Bioeng. 1997, 53 (1), 49− 57. (13) Kelley, B. D.; Booth, J.; Tannatt, M.; Wu, Q.-L.; Ladner, R.; Yu, J.; Potter, D.; Ley, A. Isolation of a peptide ligand for affinity purification of

AUTHOR INFORMATION

Corresponding Authors

*Tel: +862227404981; Fax: +862227403389; E-mail address: [email protected] (Y.S.). *E-mail address: [email protected] (L.Z.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Natural Science Foundation of China (No. 21236005), the Natural Science Foundation of Tianjin (13JCZDJC31100), the Key Technologies R&D Program of International Cooperation of Tianjin, China (11ZCGHHZ00600), the Specialized Research Fund for the 8507

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factor VIII using phage display. J. Chromatogr., A 2004, 1038 (1), 121− 130. (14) Huang, H.-J.; Yu, H. W.; Chen, C.-Y.; Hsu, C.-H.; Chen, H.-Y.; Lee, K.-J.; Tsai, F.-J.; Chen, C. Y.-C. Current developments of computeraided drug design. J. Taiwan. Inst. Chem. E 2010, 41 (6), 623−635. (15) Wilson, G. L.; Lill, M. A. Integrating structure-based and ligandbased approaches for computational drug design. Future Med. Chem. 2011, 3 (6), 735−750. (16) Liu, F.-F.; Wang, T.; Dong, X.-Y.; Sun, Y. Rational design of affinity peptide ligand by flexible docking simulation. J. Chromatogr., A 2007, 1146 (1), 41−50. (17) Liu, F.-F.; Dong, X.-Y.; Wang, T.; Sun, Y. Rational design of peptide ligand for affinity chromatography of tissue-type plasminogen activator by the combination of docking and molecular dynamics simulations. J. Chromatogr., A 2007, 1175 (2), 249−258. (18) Zhang, L.; Sun, Y. Biomimetic design of platelet adhesion inhibitors to block integrin α2β1-collagen interactions: I. Construction of affinity binding model. Langmuir 30 (16), 4725−4733. (19) Zhang, L.; Zhang, C.; Sun, Y. Biomimetic design of platelet adhesion inhibitors to block integrin α2β1-collagen interactions: II. Inhibitor library, screening and experimental validation. Langmuir 2014, 30 (16), 4734−4742. (20) Delos, S. E.; Cripe, T. P.; Leavitt, A. D.; Greisman, H.; Garcea, R. L. Expression of the polyomavirus minor capsid proteins VP2 and VP3 in Escherichia coli: In vitro interactions with recombinant VP1 capsomeres. J. Virol. 1995, 69 (12), 7734. (21) Cai, X.; Chang, D.; Rottinghaus, S.; Consigli, R. A. Expression and purification of recombinant polyomavirus VP2 protein and its interactions with polyomavirus proteins. J. Virol. 1994, 68 (11), 7609. (22) Chen, X. S.; Stehle, T.; Harrison, S. C. Interaction of polyomavirus internal protein VP2 with the major capsid protein VP1 and implications for participation of VP2 in viral entry. EMBO J. 1998, 17 (12), 3233−3240. (23) Xue, Q.; Zhang, J.-L.; Zheng, Q.-C.; Cui, Y.-L.; Chen, L.; Chu, W.T.; Zhang, H.-X. Exploring the Molecular Basis of dsRNA Recognition by Mss116p Using Molecular Dynamics Simulations and Free-Energy Calculations. Langmuir 2013, 29 (35), 11135−11144. (24) Zheng, J.; Yu, X.; Wang, J.; Yang, J.-C.; Wang, Q. Molecular modeling of two distinct triangular oligomers in amyloid β-protein. J. Phys. Chem. B 2009, 114 (1), 463−470. (25) Trott, O.; Olson, A. J. AutoDock Vina: Improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J. Comput. Chem. 2010, 31 (2), 455−461. (26) London, N.; Raveh, B.; Cohen, E.; Fathi, G.; Schueler-Furman, O. Rosetta FlexPepDock web serverHigh resolution modeling of peptide−protein interactions. Nucleic Acids Res. 2011, 39 (suppl 2), W249−W253. (27) Zhou, J.; Zheng, J.; Jiang, S. Molecular simulation studies of the orientation and conformation of cytochrome c adsorbed on selfassembled monolayers. J. Phys. Chem. B 2004, 108 (45), 17418−17424. (28) Zheng, J.; Jang, H.; Ma, B.; Nussinov, R. Annular structures as intermediates in fibril formation of Alzheimer Aβ17− 42. J. Phys. Chem. B 2008, 112 (22), 6856−6865. (29) Yu, X.; Zheng, J. Polymorphic structures of Alzheimer’s β-amyloid globulomers. PloS One 2011, 6 (6), e20575. (30) Zhao, J.; Wang, Q.; Liang, G.; Zheng, J. Molecular dynamics simulations of low-ordered Alzheimer β-amyloid oligomers from dimer to hexamer on self-assembled monolayers. Langmuir. 2011, 27 (24), 14876−14887. (31) Feng, H.; Becker, K. E.; Zhou, J.; Fichthorn, K. A. Molecular thin films on solid surfaces: Mechanisms of Melting. Langmuir 2012, 28 (19), 7382−7392. (32) Yu, X.; Wang, Q.; Lin, Y.; Zhao, J.; Zhao, C.; Zheng, J. Structure, orientation, and surface interaction of Alzheimer amyloid-β peptides on the graphite. Langmuir 2012, 28 (16), 6595−6605. (33) Lindahl, E.; Hess, B.; Van Der Spoel, D. GROMACS 3.0: A package for molecular simulation and trajectory analysis. J. Mol. Model. 2001, 7 (8), 306−317.

(34) Bussi, G.; Donadio, D.; Parrinello, M. Canonical sampling through velocity rescaling. J. Chem. Phys. 2007, 126 (1), 014101. (35) Darden, T.; York, D.; Pedersen, L. Particle mesh Ewald: An N· log(N) method for Ewald sums in large systems. J. Chem. Phys. 1993, 98 (12), 10089−10092. (36) Hess, B.; Bekker, H.; Berendsen, H. J.; Fraaije, J. G. LINCS: A linear constraint solver for molecular simulations. J. Comput. Chem. 1997, 18 (12), 1463−1472. (37) Li, J.; Zhang, L.; Sun, Y. Molecular basis of the initial platelet adhesion in arterial thrombosis: Molecular dynamics simulations. J. Mol. Graphics Modell. 2012, 37, 49−58. (38) Brooks, B. R.; Brooks, C. L.; MacKerell, A. D.; Nilsson, L.; Petrella, R. J.; Roux, B.; Won, Y.; Archontis, G.; Bartels, C.; Boresch, S. CHARMM: The biomolecular simulation program. J. Comput. Chem. 2009, 30 (10), 1545−1614. (39) Joshi, M.; Ebalunode, J. O.; Briggs, J. M. Computational insights into the interaction of the anthrax lethal factor with the N-terminal region of its substrates. Proteins: Struct., Funct., Bioinf. 2009, 75 (2), 323− 335. (40) Gorfe, A. A.; Jelesarov, I. Energetics of sequence-specific protein−-DNA association: Computational analysis of integrase Tn916 binding to its target DNA. Biochemistry. 2003, 42 (40), 11568−11576. (41) Sanner, M. F. Python: A programming language for software integration and development. J. Mol. Graphics Modell. 1999, 17 (1), 57− 61. (42) Das, R.; Baker, D. Macromolecular modeling with Rosetta. Annu. Rev. Biochem. 2008, 77, 363−382. (43) Huang, B.; Liu, F.-F.; Dong, X.-Y.; Sun, Y. Molecular mechanism of the affinity interactions between protein A and human immunoglobulin G1 revealed by molecular simulations. J. Phys. Chem. B 2011, 115 (14), 4168−4176. (44) Lafont, V.; Schaefer, M.; Stote, R. H.; Altschuh, D.; Dejaegere, A. Protein−protein recognition and interaction hot spots in an antigen− antibody complex: Free energy decomposition identifies “efficient amino acids”. Proteins: Struct., Funct., Bioinf. 2007, 67 (2), 418−434. (45) Nakanishi, A.; Nakamura, A.; Liddington, R.; Kasamatsu, H. Identification of amino acid residues within simian virus 40 capsid proteins Vp1, Vp2, and Vp3 that are required for their interaction and for viral infection. J. Virol. 2006, 80 (18), 8891. (46) Brady, J. N.; Consigli, R. A. Chromatographic separation of the polyoma virus proteins and renaturation of the isolated VP1 major capsid protein. J. Virol. 1978, 27 (2), 436−442.

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